Kohl L, Callens M, Wierenga R K, Opperdoes F R, Michels P A
Research Unit for Tropical Diseases, International Institute of Cellular and Molecular Pathology, Brussels, Belgium.
Eur J Biochem. 1994 Mar 1;220(2):331-8. doi: 10.1111/j.1432-1033.1994.tb18629.x.
The gene of triose-phosphate isomerase in Leishmania mexicana has been cloned and characterized. The gene encodes a polypeptide of 251 amino acids, with a calculated molecular mass of 27,561 Da and a net charge of +2. Only one gene could be detected, although the enzyme is present in two different compartments of the cell, in microbody-like organelles called glycosomes and in the cytosol. The primary structure of the enzyme has many features in common with that of triose-phosphate isomerase in the related organism Trypanosoma brucei. Their sequences are 68% identical. The residues constituting the subunit interface are highly conserved between the enzyme of L. mexicana and T. brucei, but are mostly different from those in the enzyme of other organisms. One major substitution was detected in the interface region of the L. mexicana protein: a glutamate was found at position 66, instead of glutamine in all other available 20 sequences. The glutamine is thought to be important for the stability of the dimeric enzyme. L. mexicana triose-phosphate isomerase has been overexpressed in Escherichia coli. Growth conditions were established to obtain high levels of soluble and active protein. The enzyme has been purified to near homogeneity. It appears a stable dimeric protein with a specific activity of 5500 units/mg protein, a subunit mass of 28 kDa and an isoelectric point of 9.0. The enzyme has also been partially purified from glycosomes of cultured L. mexicana promastigotes. Some kinetic properties of the recombinant protein have been compared with those of the promastigote enzyme and with the values previously reported for the T. brucei enzyme. The kinetics of the different enzyme preparations were very similar. For the recombinant enzyme the following values were measured: with glyceraldehyde 3-phosphate as substrate Km = 0.30 +/- 0.05 mM and kcat = 2.5 x 10(5) min-1; with dihydroxyacetone phosphate as substrate Km = 1.3 +/- 0.3 mM and kcat = 2.8 x 10(4) min-1.
墨西哥利什曼原虫的磷酸丙糖异构酶基因已被克隆并进行了表征。该基因编码一个由251个氨基酸组成的多肽，计算分子量为27,561道尔顿，净电荷为+2。尽管该酶存在于细胞的两个不同区室中，即在称为糖体的微体样细胞器和胞质溶胶中，但只能检测到一个基因。该酶的一级结构与相关生物体布氏锥虫中的磷酸丙糖异构酶有许多共同特征。它们的序列有68%相同。构成亚基界面的残基在墨西哥利什曼原虫和布氏锥虫的酶之间高度保守，但与其他生物体的酶中的残基大多不同。在墨西哥利什曼原虫蛋白的界面区域检测到一个主要替代：在位置66处发现一个谷氨酸，而在所有其他可用的20个序列中为谷氨酰胺。谷氨酰胺被认为对二聚体酶的稳定性很重要。墨西哥利什曼原虫磷酸丙糖异构酶已在大肠杆菌中过表达。建立了生长条件以获得高水平的可溶性和活性蛋白。该酶已被纯化至接近均一。它似乎是一种稳定的二聚体蛋白，比活性为5500单位/毫克蛋白，亚基质量为28 kDa，等电点为9.0。该酶也已从培养的墨西哥利什曼原虫前鞭毛体的糖体中部分纯化。已将重组蛋白的一些动力学特性与前鞭毛体酶的动力学特性以及先前报道的布氏锥虫酶的值进行了比较。不同酶制剂的动力学非常相似。对于重组酶，测量了以下值：以3-磷酸甘油醛为底物时，Km = 0.30±0.05 mM，kcat = 2.5×10⁵ min⁻¹；以磷酸二羟丙酮为底物时，Km = 1.3±0.3 mM，kcat = 2.8×10⁴ min⁻¹。
